Diagnosis and treatment methods for entry of gastrointestinal contents into respiratory tract

ABSTRACT

A method of diagnosing in a subject for the purpose of determining if the subject&#39;s gastrointestinal contents has entered the subject&#39;s respiratory tract. The qualitative analysis can be also expanded into quantitative analysis, enabling the estimation of either the concentration, or the amount, or both, of the gastrointestinal contents that entered the respiratory tract. The invention also provides methods of treatment based on the identification of aspiration using the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Ser. No.61/777,933, filed Mar. 12, 2013, U.S. Provisional Ser. No. 61/777, 955,filed Mar. 12, 2013, and is a Continuation-In-Part of U.S. Ser. No.13/162,344, filed Jun. 16, 2011 which claims priority to U.S.Provisional Ser. No. 61/355,859, filed Jun. 17, 2010, and U.S.Provisional Ser. No. 61/370,588, filed Aug. 4, 2010, all of which areincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to the field of medical diagnoses andmore particularly to methods and materials used for the detection ofentry of gastrointestinal contents into the respiratory tract, withspecial attention to the diagnosis for the purpose of prevention andtreatment of disease.

BACKGROUND OF THE INVENTION

Gastro esophageal reflux disease (GERD) is a condition in which some ofthe stomach contents (solid and/or liquid) move backwards from thestomach into the esophagus (the tube from the mouth to the stomach).This action can irritate the esophagus, causing heartburn and othersymptoms.

Gastroesophageal reflux disease (GERD), gastro-oesophageal refluxdisease (GORD), gastric reflux disease, or acid reflux disease isdefined as chronic symptoms or mucosal damage produced by the abnormalreflux of stomach acid to the esophagus. A typical symptom is heartburn.

This is commonly due to transient or permanent changes in the barrierbetween the esophagus and the stomach. This can be due to incompetenceof the lower esophageal sphincter, transient lower esophageal sphincterrelaxation, impaired expulsion of gastric reflux from the esophagus, ora hiatal hernia.

A different type of acid reflux which produces respiratory and laryngealmanifestations is laryngopharyngeal reflux (LPR), also calledextraesophageal reflux disease (EERD). Unlike GERD, LPR is unlikely toproduce heartburn, and is thus sometimes called silent reflux.

The gastrointestinal contents may thus also enter the respiratory tractas a result of any condition that causes the backward movement of thegastrointestinal contents from the stomach to esophagus. Thegastrointestinal contents contain many substances that are likely to beharmful to the respiratory tract: acid, digestive enzymes,microorganisms, allergens, proinflammatory subtances and so on. There isincreasing evidence that gastro esophageal reflux disease (GERD) is theunderlying mechanism behind many disease conditions of the respiratorytract, such as infections and high morbidity in subjects with lungtransplants, asthma, bronchitis, bronchiectasis, pulmonary fibrosis andso on. At present, there are no acceptable methods to detect the entryof gastrointestinal (GI) contents into the respiratory tract.

SUMMARY OF THE INVENTION

Diagnostic formulations for use in diagnosing and treating entry ofgastrointestinal contents into the respiratory tract (“aspiration”) insubjects, with special attention to the diagnosis for the purpose ofprevention and treatment of diseases associated with aspiration. Thesubjects that may be diagnosed and/or treated using such methods areanimals, prefereably mammals, including humans.

In a first embodiment, the concentration of the gastrointestinalcontents entering the respiratory tract can be estimated by adding adetectable non-toxic label that is not absorbed from thegastrointestinal tract or from the respiratory tract. The label shouldbe in a form that is biocompatible with the respiratory tract and thegastrointestinal tract. The label also should not be destroyed in thegastrointestinal or respiratory tract. If the contents of thegastrointestinal tract enters the respiratory tract, the respiratoryfluid can be sampled (e.g., by bronchoscopy) and the concentration ofthe label in the respiratory tract can be measured, thus estimating theconcentration of the gastrointestinal contents that entered therespiratory tract.

For example, the diagnostic formulation may be comprised of aningestible liquid; and a plurality of particles comprised of abiocompatible polymer such as carnauba wax and a non-radioactive labelsuch as fluorescein.

Accordingly, a first embodiment provides a method of diagnosingrespiratory fluid in a subject, comprising orally administering to asubject a diagnostic formulation comprising a plurality of particles,wherein the particles comprise a biocompatible material that is notdestroyed in the gastrointestinal or respiratory tracts and a detectablelabel, allowing the formulation to remain in the subject over a periodof time during which the subject would be expected to aspirate theformulation from the gastrointestinal tract into the respiratory tract,accessing respiratory fluid from the subject, and analyzing therespiratory fluid to determine if the fluid contains the detectablelabel. The formulation optionally comprises an aqueous carrier.

In certain aspects, the biocompatible material comprises carnauba wax.In other aspects, the biocompatible material is associated with adetectable label, e.g. a fluorescent label, a radioactive label, amagnetic label and a UV label. In a specific aspect, the detectablelabel is an optically fluorescent label, e.g. fluorescein. The label ispreferably encapsulated in a material which is not degraded in therespiratory and gastrointestinal tracts. In a specific aspect, the labelis encapsulated in carnauba wax.

The the respiratory fluid n the methods of the invention used isaccessed in any manner that allows analysis of fluid from therespiratory tract of the subject. In preferred aspects, the body fluidanalyzed is fluid collected, e.g., using bronchoscopy, spontaneoussputum collection or induced sputum production. In some aspects, theanalyzing of the detectable label in the collected respiratory fluidcomprises determining a number of labels per unit volume of respiratoryfluid obtained from the subject.

In certain embodiments, the methods of the invention further comprisethe administration of a control formulation to a subject to establish alevel of the agent for analysis of the presence and/or concentration ofgastric contents in the respiratory fluid of a subject either before orfollowing administration of the diagnostic formulation. The controlformulation may be administered by inhalation, e.g., to establish levelsof an agent that may be present should the agent be aspirated. Thecontrol formulation also may be administered by ingestion to establish abaseline for the agent should it not be aspirated. For the latteraspect, the control formulation is preferably administered during aperiod of time when the subject is not expected to experience aspirationof gastrointestinal contents into the respiratory tract.

In a specific aspect, detection of the level of the agent or detectablelabel indicative of aspiration of gastrointestinal contents into therespiratory tract requiring medical intervention is based on acomparison of the level observed following administration of thediagnostic formulation and the level observed following administrationof the control formulation. In the case of administration of the controlformulation to establish a negative baseline, the level of agent ordetectable label determined for administering to the subject a medicaltreatment to reduce or prevent such aspiration is determined as anincreased level of agent over that seen following the controladministration. In the case of the control administration establishingthe level in a bodily fluid, the level of agent or detectable labeldetermined for administering to the subject a medical treatment toreduce or prevent such aspiration is determined by comparison to thelevels produced using the control formulation.

In another aspect, detection of the level of the agent or detectablelabel indicative of aspiration of gastrointestinal contents into therespiratory tract requiring medical intervention is based on rangesestablished through clinical practice. This can be based on the levelsof aspiration detected in a range of previous patients or a range basedon a simlulation of predicted values of agent and/or detectable label.

In specific aspects, the invention provides a method of diagnosingrespiratory fluid in a subject suffering from gastroesophageal refluxdisease (GERD), comprising orally administering to a subject suspectedof suffering from gastro esophageal reflux disease (GERD) a formulationcomprised of a plurality of particles comprised of fluorescein andcarnauba wax. allowing the formulation to remain in the subject over aperiod of time during which the subject would be expected to aspiratethe formulation from the gastrointestinal tract into the respiratorytract, accessing respiratory fluid from the subject, and analyzing therespiratory fluid to determine the presence of fluorescein, therebdetermining if the subject aspirates gastrointestinal contents into therespiratory tract.

The invention further comprises a method of determining the respiratoryfluid contains a concentration of gastric contents indicative ofaspiration of gastrointestinal contents into the respiratory tract andadministering to the subject a medical treatment to reduce or preventsuch aspirations. Such medical treatments can include pharmacologicalintervention or surgical intervention.

In specific embodiments, the invention provides a diagnosticformulation, comprising an ingestible liquid carrier; and a plurality ofparticles comprised of carnauba wax and a detectable label. Theformulation prefereably comprises an aqueous carrier. The detectablelabe can be, e.g., a fluorescent label, a radioactive label, a magneticlabel and a UV label. In a specific aspect, the particles comprisefluorescein encapsulated in carnauba wax. A preferred label isflourescein. In another specific aspect, the particles comprise aradioactive material encapsulated in carnauba wax. In a preferredaspect, the label is encapsulated in a material which is not degraded inthe respiratory and gastrointestinal tracts.

In other embodiments, the invention provides a method of treating asubject suffering from entry of gastrointestinal contents orallyadministering to a subject a formulation comprising a plurality ofparticles, wherein the particles comprise a biocompatible material thatis not destroyed in the gastrointestinal or respiratory tracts and adetectable label, allowing the formulation to remain in the subject overa period of time during which the subject would be expected to aspiratethe formulation from the gastrointestinal tract into the respiratorytract; accessing respiratory fluid from the subject, and analyzing therespiratory fluid to determine the concentration of detectable label inthe respiratory fluid; determining if the concentration of detectablelabel in the respiratory fluid is indicative of aspiration ofgastrointestinal contents into the respiratory tract requiringpharmacological treatment, and administering to the subject apharmacological treatment to reduce or prevent such aspirations.

In preferred aspects, the the respiratory fluid is accessed bycollecting fluid from lungs of the subject. In certain aspects, theanalyzing comprises determining a number of fluorescein particles perunit volume of respiratory fluid obtained from the subject.

In certain embodiments, the invention provides for dual use of an agentthat is not absorbed from the gastrointestinal tract of a subject but isabsorbed from the respiratory tract for estimating the amount of thegastrointestinal contents that entered the respiratory tract, andparticles of a biocompatible material that is not destroyed in thegastrointestinal or respiratory tracts and a detectable label toestimate the concentration of gastrointestinal contents that entered therespiratory tract. In certain aspects, these agents and particles can beused in the same formulation. The administered agent could then bedetected using a bodily fluid (e.g., blood, a blood product or urine)and the particles detected in, e.g., the sputum or bronchial fluid of asubject. Using both approaches of the method of the invention will allowthe determination of both the amount of gastric contents that hasaspirated into a subject's respiratory system, and the concentration ofthe gastric contents in the bodily fluid.

For example, a formulation containing both particles oflabel-encapsulated carnauba wax and an agent such as a cromolyn salt maybe administered to a subject. Following administration, the analysis ofthe cromolyn salt in the subject, e.g., through analysis of blood orurine, can be used to determine the amount of gastric contents aspiratedinto the respiratory system while the analysis of the detectable labelin the bodily fluid of the subject, e.g. through detection of the labelin sputum, can determine the concentration of the gastric contents inthe bodily fluid.

The present invention also provides a method of determining the amountand concentration of gastric contents in the respiratory fluid of asubject, comprising orally administering to a subject a formulationcomprising a plurality of particles, wherein the particles comprise abiocompatible material that is not destroyed in the gastrointestinal orrespiratory tracts and a detectable label, and an agent that is notabsorbed from the gastrointestinal tract of a subject but is absorbedfrom the respiratory tract of the subject, allowing the formulation toremain in the subject over a period of time during which the subjectwould be expected to aspirate the formulation from the gastrointestinaltract into the respiratory tract, accessing the respiratory fluid fromthe subject, analyzing the respiratory fluid to determine theconcentration of the detectable label in the respiratory fluid,accessing a body fluid other than respiratory fluid from the subject,and analyzing the body fluid to determine if the fluid contains a levelof the agent indicative of the amount of aspiration of gastrointestinalcontents into the respiratory tract. The body fluid other thanrespiratory fluid can be urine, blood or blood products.

The invention also futher provides determining the respiratory fluidcontains a concentration of gastric contents in the respiratory tractindicative of aspiration of gastrointestinal contents into therespiratory tract, and administering to the subject a medical treatmentto reduce or prevent such aspirations. Such medical treatments caninclude pharmacological intervention or surgical intervention.

Alternatively, the diagnostic methods of the invention may employ anagent that is not absorbed from the gastrointestinal tract, but isabsorbed from the respiratory tract. Levels of this agent in a subject'sbodily fluid, e.g., blood, plasma, serum or urine, can be detected andused to estimate the amount of the gastrointestinal contents thatentered the respiratory tract. This is done by measuring the amount ofthe agent that has entered the circulation (from the respiratory tract)by taking samples of blood, plasma, serum or urine, and quantifying theamount of the label in those fluids. If the drug is excreted also intosaliva, then the saliva samples may be the most convenient method. Forexample, cromolyn sodium (sodium cromoglycate) is a harmless substancethat is soluble in water, is not destroyed in the gastrointestinal tractor the respiratory tract, is not absorbed from the gastrointestinaltract but is absorbed from the respiratory tract. A water solution ofcromolyn sodium can therefore be swallowed and the concentrations ofcromolyn in blood and urine samples can be used to estimate the amountof gastrointestinal contents that entered the respiratory tract.Cromolynic acid and other salts of cromolyn can be used instead ofsodium cromolyn, or substances that are structurally similar such asnedocromil sodium. Another class of substances that have members thatare poorly absorbed from the gastrointestinal tract but are wellabsorbed from the lung are anticholinergic drugs also known asmuscarinic acid receptor antagonists such as tiotropium bromide.

Accordingly, the invention provides a method of diagnosing respiratoryfluid in a subject, comprising orally administering to a subject adiagnostic formulation comprising an agent that is not absorbed from thegastrointestinal tract of a mammal but is absorbed from the respiratorytract of a mammal, allowing the formulation to remain in the subjectover a period of time during which the subject would be expected toaspirate the formulation from the gastrointestinal tract into therespiratory tract, accessing a body fluid from the subject, andanalyzing the body fluid to determine if the fluid contains a level ofthe agent indicative of aspiration of gastrointestinal contents into therespiratory tract.

In a more specific aspect, the invention provides a method of diagnosingrespiratory fluid in a subject, comprising orally administering to asubject a diagnostic formulation comprising a cromolyn salt, allowingthe diagnostic formulation to remain in the subject over a period oftime during which the subject would be expected to aspirate theformulation from the gastrointestinal tract into the respiratory tract,accessing a body fluid from the subject; and analyzing the body fluid todetermine if the fluid contains a level of the cromolyn salt indicativeof aspiration of gastrointestinal contents into the respiratory tract.

The invention also provides methods of treating subjects in need ofmedical intervention due to aspiration of gastic contents intorepiratory fluid. Accordingly, the invention provides a method oftreating a subject suffering from entry of gastrointestinal contentsinto the respiratory tract, comprising orally administering to a subjecta diagnostic formulation comprising an agent that is not absorbed fromthe gastrointestinal tract of a mammal but is absorbed from therespiratory tract of a mammal, allowing the formulation to remain in thesubject over a period of time during which the subject would be expectedto aspirate the formulation from the gastrointestinal tract into therespiratory tract, accessing a body fluid from the subject wherein thebody fluid, analyzing the body fluid to determine the level of the agentin the body fluid, determining the fluid contains a level of the agentsalt indicative of aspiration of gastrointestinal contents into therespiratory tract, and administering to the subject a pharmacological orsurgical treatment to reduce or prevent such aspirations, or advisingthe subject to change their diet, timing of meals, body posture at nightand so on to minimize or prevent the aspiration of gastrointestinalfluid into the respiratory tract. One or both of the diagnostic agengs,such as fluorescein encapsulated in carnauba wax particles or cromolynsolution, either individually, or as a mixture, can be subsequentlyadministered to probe the effectiveness of the intervention to treat thecondition of aspiration of gastro-intestinal fluid into the respiratorytract.

The invention also provides a method of treatment, comprising detectinga level of cromolyn salt indicative of aspiration of gastrointestinalcontents into the respiratory tract, and administering to the subject apharmacological or surgical treatment, or other interventions to reduceor prevent such aspiration. The invention also provides a methodtreating a subject suffering from entry of gastrointestinal contentsinto the respiratory tract, comprising orally administering to a subjecta diagnostic formulation comprising a cromolyn salt allowing theformulation to remain in the subject over a period of time during whichthe subject would be expected to aspirate the formulation from thegastrointestinal tract into the respiratory tract, accessing a bodyfluid from the subject, and analyzing the body fluid to determine thelevel of cromolyn salt in the body fluid, determining the fluid containsa level of cromolyn salt indicative of aspiration of gastrointestinalcontents into the respiratory tract, and administering to the subject apharmacological or surgical treatment to or other intervention to reduceor prevent such aspirations.

The invention also provides administering a control formulationcomprising the agent used in the diagnostic formulation to the subjectat an occasion different to that when the orally administered diagnosticformulation is administered, accessing a body fluid from the subjectfollowing the administration of the control formulation, and analyzingthe body fluid to determine if the fluid contains the agent.

The control formulation can be administered orally or by inhalation.Preferably, the body fluid analyzed is collected following theadministration of the control formulation and prior to analyzing thebody fluid. In some aspects, the control formulation is administeredduring a period of time when the subject is not expected to experienceaspiration of gastrointestinal contents into the respiratory tract. Inother aspects, the control formulation is administered during a periodof time when the subject is expected to experience aspiration ofgastrointestinal contents into the respiratory tract. In yet otheraspects, the control formulation is a positive control administered todetermine the amount of agent expected to be detected in a bodily fluidshould aspiration occur.

A particular aspect of the invention involves the use of dipsticktechnology in order to assay for the presence of an agent such ascromolyn in a body fluid such as urine or saliva. When the dipstickindicates the presence of the agent such as cromolyn the presence ofsuch is a positive result indicating that the subject is aspiratingcontents of the gastrointestinal tract into the respiratory tract of thesubject. Those skilled in the art will appreciate that a wide range ofdifferent embodiments of dipstick technologies can be used in connectionwith the invention. Examples of such dipstick assays are disclosedwithin U.S. Pat. No. 5,256,372 issued Oct. 26, 1993; U.S. Pat. No.4,968,604 issued Nov. 9, 1990; and U.S. Pat. No. 7,972,837 issued Jul.5, 2011, all of which are incorporated herein by reference as are thepatents and publications cited within these patents.

The dipstick assay methodology generally requires the presence of apaper or cardboard substrate which is dipped into a solution to betested. The substrate allows for the solution to migrate upward bycapillary action. Although different embodiments are possible, thesubstrate generally includes a reagent that will interact with cromolynso that an observable effect is generated to detect cromolyn at leastqualitatively, and preferably quantitatively. One example is that thedipstick substrate contains a reagent that reacts with the agent such ascromolyn, where the reaction gives rise to a distinct color which can bereadily detected by an unaided human eye under normal room lighting. Theintensity of this color is the measure of the amount of the agent insolution. Some intermediate steps may also be required to cause andenhance the formation of the color reaction. Such chemistry wasdescribed for example by K Görlitzer, G Badia, P G Jones. Pharmazie.2001 May; 56 (5):401-6.

Another method uses an antibody which binds to a particular agent suchas cromolyn with respect to the present invention. The substrate mayinclude an anti-antibody which binds to the antibody that binds theagents such as cromolyn. Further, the antibody or anti-antibodygenerally includes a visually detectable label wherein the label can bereadily detected by an unaided human eye under normal room lighting. Thesystem may show a negative response when a signal or line across thepaper appears at a particular location and a positive response when asignal such as a line appears at a different location on a substratepaper.

Certain devices can be used in the methods of the invention. Multipleforms of dipstick devices may be used to carry out the presentinvention.

In a first aspect, the dipstick device for the detection of an agentindicative of aspiration of gastrointestinal contents into therespiratory tract of a subject, comprising a substrate comprised ofpaper a first antibody which binds to an agent that is not absorbed fromthe gastrointestinal tract of a subject but is absorbed from therespiratory tract of a subject, a second antibody which binds to thefirst antibody; and a visually detectable label.

In another aspect, the dipstick device for the detection of an agentindicative of aspiration of gastrointestinal contents into therespiratory tract of a subject comprises a substrate comprised of paperand a first reagent that reacts with the agent to form a second reagentoptically distinguishable from the first reagent. In a specific aspect,the first agent is a colored dye and the second reagent is a colordistinguishable from the color of the first agent.

In another aspect, the dipstick device for the detection of an agentindicative of aspiration of gastrointestinal contents into therespiratory tract of a subject comprises a substrate comprised of paperand a first reagent that reacts with the agent, and a second reagentthat enables the reaction of the first reagent to form a dye with theagent that is of a different color than the color either the first orthe second reagent.

In another aspect, the dipstick device for the detection of an agentindicative of aspiration of gastrointestinal contents into therespiratory tract of a subject comprises a substrate comprised of paperand a reagent that reacts with the agent to form a compound with avisually detectable color.

In another aspect, the dipstick device for the detection of an agentindicative of aspiration of gastrointestinal contents into therespiratory tract of a subject comprises a substrate comprised of paperand a first reagent that reacts with the agent and a second reagent thatenables the reaction of the first reagent to form a compound with avisually detectable color.

The dipstick devices of the invention generally detect a specific agent,e.g., cromolyn salts, cromolynic acid, nedocromil, nedocromil salts andmuscarinic acid receptor antagonists.

Those skilled in the art will know that other methods of detection canbe used based on various properties of the specific agents. Portableelectronic technology devices including cellphones, smartphones, tabletsand so on can be used to detect unique optical properties of thediagnostic agents in the collected body fluids. The subject beingdiagnosed can read the result on the electronic device, or the signalcan be transmitted to a specialized center for analysis, or to ahealthcare professional.

Both the agent and the particle approaches can be also used purely forqualitative purposes to detect the entry of gastrointestinal contentsinto the respiratory tract. A label that is destroyed neither in therespiratory tract, nor in the gastrointestinal tract and which also hasthe properties of being absorbed from the respiratory tract but not fromthe gastrointestinal tract, can be used for qualitative detection of theentry of gastrointestinal contents into the respiratory tract. Such alabel is swallowed and its presence is detected in the blood samples, orin urine if it is excreted via kidneys from blood to urine.Alternatively, a label that is not absorbed or destroyed in thegastrointestinal and respiratory tracts can be swallowed andsubsequently detected in the respiratory fluid if the subject suffersfrom a condition that moves the contents of the gastrointestinal tractinto the respiratory tract.

Although GERD is generally referred to here, those skilled in the artwill understand that this invention is applicable to use in connectionwith any disorder that causes entry of gastrointestinal contents intothe respiratory tract of the subject. Different subjects may besuffering from different disorders which result in involuntaryaspiration of gastrointestinal contents. This may result in some of thegastrointestinal contents entering the respiratory tract of the subjectcausing damage. The present invention is intended to detect the presenceof such gastrointestinal contents in the respiratory tract regardless ofa particular disease or disorder which may have resulted in the presenceof the gastrointestinal contents in the respiratory tract. The inventionalso describes medical interventions, e.g., surgical or pharmacologicalinterventions, to prevent the entry of gastrointestinal tract contentsinto the respiratory tract.

A method of diagnosing respiratory fluid in a subject suffering fromgastro esophageal reflux disease (GERD) is disclosed. The methodcomprises (1) orally administering to a subject suspected of sufferingfrom aspiration of gastrointestinal fluid into the respiratory tract,e.g., subjects at high risk who have gastro esophageal reflux disease(GERD), or subjects with respiratory condition of unknown origin, aformulation comprised of a plurality of particles comprised of abiocompatible material (e.g. carnauba wax) and a detectable label (e.g.a fluorescent label), (2) allowing the formulation to remain in thesubject over a period of time during which the subject would be expectedto regurgitate formulation, (3) collecting respiratory fluid from thesubject, and (4) analyzing the respiratory fluid to determine if thefluid contains the detectable label, and thereby determining if thesubject aspirated gastrointestinal contents into the respiratory tract.

Those skilled in the art will know that there are numerous ways in whichthe label contained within the material of the particles that aredigested neither in the gastrointestinal tract, nor in the respiratorytract, can be detected. External counters can detect the presence ofradiolabel in such particles in the respiratory fluid. Alternatively,magnetic detectors can be used for magnetic labels, or the magneticparticles can be extracted with a magnet and their quantity determined,e.g., by weighing or counting them. The preferred embodiment is to usefluorescein in the label contained in carnauba wax particles. Therespiratory fluid can be heated to melt the carnauba wax and thefluorescein released from the particles can then be detected using itsfluorescent properties. Other extraction methods that incorporate theuse of organic solvents to dissolve or extract the carnauba wax can beemployed.

By carrying out the steps as described above it is possible to analyzethe fluid collected from the respiratory tract and determine theconcentration of the label in the respiratory fluid. The higher theconcentration of the label in the respiratory fluid the greater thepotential damage to the subject's respiratory tract due to involuntaryaspiration of gastrointestinal contents. Higher concentrations of thelabel may suggest more aggressive treatment of the subject.

In another embodiment of the invention, the formulation is also orallyadministered to the subject. However, instead of comprising particleswith a fluorescent label (or other label) coated with carnauba wax (orother material) the formulation comprises a substance (an agent) such ascromolyn sodium. Cromolyn sodium is water soluble and harmless. Further,cromolyn sodium is not absorbed in the gastrointestinal tract, but isabsorbed from the respiratory tract into the blood stream. Those skilledin the art will recognize other compounds which have thesecharacteristics. However, it is important to select compounds that areknown not to affect in that particular subject, or in that group ofsubjects, the aspiration of gastrointestinal contentss into therespiratory tract. When the formulation of such an agent that is notcausing or preventing aspirations is orally administered to the subjectand there is aspiration of gastrointestinal contents into therespiratory tract, it is possible to test for the presence of the agentin the subject's blood or urine.

Thus, in accordance with this embodiment of the method the respiratoryfluids of the subject does not need to be extracted. This method allowsfor theestimation of the total amount of gastrointestinal contents whichhas entered the respiratory tract.

An example of a substance that is safe to both the respiratory tract andthe gastrointestinal tract is cromolyn sodium. Cromolyn is in fact adrug with very few adverse reactions. Cromolyn sodium is thought to actby inhibiting of the release of histamine and leukotrienes (SRS-A) fromthe mast cell. It is therefore possible that in some subjects the causeof aspiration of the gastrointestinal tract contents into therespiratory tract is inhibited by the action of cromolyn sodium andsimilar drugs. This desirable pharmacological effect can be detected forexample by administering orally to the subject on one occasion carnaubawax particles coated with fluorescein alone and on another occasionthese particles together with cromolyn sodium. If on the first occasionthe carnauba wax particles are detected in the respiratory tract fluidssuch as the sputum from the subject but they are not detected when suchparticles are co-administered with cromolyn, then this suggests thatcromolyn is minimizing or blocking the aspiration of gastrointestinalcontents into the respiratory tract. Of course, those skilled in the artwill know that other types of biocompatible, non-reactive substances canbe used instead of carnauba wax, fluorescein or cromolyn sodium.

When a substance such as a cromolyn sodium is administered to thesubject it may be advisable to first administer a control formulation ofcromolyn sodium orally to the subject in the morning. The dose is orallyadministered at a time when the subject would not be expected toexperience any reflux. After administering the formulation such ascromolyn sodium the subject is not allowed to sleep or even lie down.Further, any steps that may need to be taken in order to avoid thesubject experiencing reflux into the respiratory tract are taken. Bloodand urine samples are then taken during the day and analyzed as acontrol. It may be that no cromolyn appears in the blood or urine.However, if a small amount appears, then that level of cromolyn can beused as a baseline to compare to the level when the formulation isadministered and the subject is allowed to lie down, sleep and normallyexperience reflux into the respiratory tract. When the secondadministration occurs the same amount of cromolyn sodium is orallyadministered as was administered in the control. Carrying out acomparison will avoid false positives in the diagnosis of the subject,particularly in subjects that have abnormally high oral absorption ofcromolyn. It is also possible to maximize the probability of aspirationin a particular subject, for example with heavy meals or foods thatprovoke GERD. It is pointed out that in most “normal” subjects therewill be substantially no absorption of cromolyn into the blood or urineunless the cromolyn is absorbed via the respiratory tract.

Yet another means to establish whether the subject is likely to besuffering from the aspiration of gastrointestinal contents into therespiratory tract is to establish the amounts and concentration of thediagnostic substances in trials in which a group of subjects withaspirations documented by other methods such as video monitoring viabronchoscopy or gamma scintigraphic detection in the respiratory tractof ingested radioactive substances, are compared with the quantitiesdetected in healthy subjects who do not have such aspiration. Thresholdlevels of these diagnostic markers above which the aspiration is likelycan thus be established.

In yet another embodiment of the invention the two embodiments describedabove (coated particles and cromolyn sodium) are combined. Thus, thecomposition which is orally administered to the subject is a liquidformulation which includes the labeled particles coated with thebiocompatible material such as carnauba wax and a material such ascromolyn sodium dissolved in the surrounding aqueous carrier. Inaccordance with this method follow-up analysis involves testing therespiratory fluid for the presence of the labeled particles to determinethe concentration of the label in the fluid, and testing the bloodand/or urine of the subject in order to calculate the total amount ofgastrointestinal contents which was aspirated into the respiratorytract.

The combination method described above can, of course, also be carriedout using the control. The control is carried out as described above orthe cromolyn sodium formulation is administered to the subject purely asa control and samples of the subject's blood and/or urine are takenduring a period of time when the subject is not expected to experiencereflux, such as during the day when the subject is not lying down orsleeping.

In still another embodiment of the invention the labeled material iscoated with a composition which does not degrade inside the human bodybut which can be removed or degraded readily outside the human body bythe application of heat or other chemicals. More specifically, thematerial coating the label does not melt at body temperature may beremoved by the application of heat at a temperature above bodytemperature (>40° C.) or by the application of compound which readilydissolves the composition coating the label.

In yet another aspect of the invention positive controls can be used.For example, the subject can be administered a cromolyn formulation byinhalation. By knowing the amount of cromolyn administered into thesubject's respiratory tract, and thereafter testing for cromolyn in thesubject's blood and urine a comparison can be made to later tests whenthe cromolyn will be absorbed from reflux out of the subject's stomachinto the respiratory tract. It is also, of course, possible to carry outboth a negative and a positive control on the same subject.

The invention includes a method of diagnosing respiratory tract fluid ina subject by first orally administering to a subject a formulationcomprised of a plurality of particles comprised of a biocompatiblematerial and a detectable label. The formulation may include any numberof particles, but for example 100, 500, 1,000 or more, 10,000 or more,100,000 or more particles. The biocompatible material may be carnaubawax or a different non-reactive biocompatible polymer and theformulation may be an aqueous carrier which may be simply water. Thelabel is preferably a non-toxic label such as a fluorescent label thatis encapsulated within the biocompatible material. The label may also bea radioactive label, a magnetic label and/or a UV labeled material.After administering the formulation the subject is allowed to restduring a period of time where aspiration of the formulation from thegastrointestinal tract into the respiratory tract would be expected tooccur. After this time respiratory fluid is extracted from the subjectsuch as by the use of bronchoscophy. Alternatively, the subject mayspontaneously produce sputum as a sample of respiratory fluid and thelabeled particles are then detected in this body fluid. Yet anotheroption is to induce sputum production by one of the methods known to beused for this purpose, such as inhalation of hypertonic saline. Therespiratory fluid collected from the subject is then analyzed in orderto determine if the fluid contains the detectable label present withinthe formulation which was orally administered. The presence of theformulation label indicates that the subject has experienced aspirationof the gastrointestinal contents into the respiratory tract.

If the diagnosis is thus made, then it may be advisable to starttreating the subject to minimize or eliminate the aspiration ofgastro-intestinal contents into the respiratory tract, or at least tominimize the adverse effects of such aspirations.

There are several types of interventions to prevent or reduceaspirations or minimize them and their effects that have been described.Medical treatments for use with the present invention includepharmacologic treatments and surgical treatments. The three main typesof medicines to treat GERD are antacids, H2RAs (histamine type 2receptor antagonists), and PPIs (proton pump inhibitors). Exemplarypharmacologic treatments that can be used with the invention includeadministration of H2Ras, e.g., Famotidine, Nizatidine, Ranitidine, orCimetidine; or proton pump inhibitors, e.g., Omeprazole, Lansoprazole,Pantoprazole, Rabeprazole, Esomeprazole, or Dexlansoprazole. Surgicaltreatments include fundoduplication and endoscopic techniques. Otherforms of intervention include assistance with the institution oflifestyle changes in the subject, including changes to a subject'ssleeping circumstances or other behavior modification (e.g., dietarychanges, changes in consumption of drugs or alcohol, and the like).

It is common for patients suspected of having recurrent GERD withaspiration simply to be treated with a proton-pump inhibitor to reducethe acidity of gastric contents. While this is effective for refluxesophagitis, it is ineffective for the consequences of aspiration of theother components of gastric contents, including food particles anddigestive enzymes. Effective prevention of microaspiration secondary toGERD would require surgical intervention, such as gastricfundoplication. At present, the impact of surgical interventions in GERDwith microaspiration is difficult to assess without a simple, directdiagnostic test. The present invention therefore combines diagnosis ofaspirations with a medical intervention such as gastric fundoplicationor pharmacological treatment. A milder intervention may be change in thetiming, quality and quantity and food and drinks for the subject,sleeping position and so on. The impact of these interventions can bemonitored using the methods described in this invention, with diagnosticsubstances such as a fluorescent marker encapsulated in small particlesmade from a non-digestable biocompatible matrix, or solutions ofsubstances such as cromolyn sodium which is chemically stable in thebody, safe and well tolerated, poorly absorbed from the gastrointestinaltract but well absorbed from the respiratory tract.

An aspect of the invention is that it is safe, and convenient for thesubject.

Another aspect of the invention is that it is easily administered evenin a primary healthcare setting, fast and cost-effective, with highspecificity and selectivity. The use of simple “dipstick” methods isparticularly attractive: the subject is given a dose of the substancesuch as cromolyn, then urine is collected from the subject. A dipstickis placed in contact with the urine sample, followed by minimummanipulation and observation of changes in the dipstick appearanceindicative of the presence and quantity of the diagnostic substance,such as cromolyn sodium.

Another aspect of the invention is that it avoids the use ofradiolabels, because they are not practicable in a routine setting andmultiple exposures to radioactivity raises safety concerns.

Another aspect of the invention is that to be able to estimate theconcentration of the gastrointestinal contents that entered therespiratory tract, it is necessary to to avoid the use of labels thatenter the blood circulation.

Another aspect of the invention is that it uses labels that stay as atracer of the GI fluids that enter into the respiratory tract and remainintact in the GI and respiratory tracts.

Another aspect of the invention is that it uses materials presented informs that are safe in the respiratory and GI tracts.

Another aspect of the invention is that it retains the label while inthe body, presents the label readily when outside the body to a detectorproviding high specificity and selectivity (i.e., only the labelmaterial that was initially swallowed or otherwise placed into the GItract and then enter the respiratory tract will be detected).

Another aspect of the invention is that the label used can be detectedeven if only minute quantities of the gastrointestinal materials enteredthe respiratory tract.

Another aspect of the invention is that the diagnostic method to detectthe presence of aspirations of gastro-intestinal contents into therespiratory tract is used in a subject and if the result is positive,treatment of the condition starts. The impact of the treatment can bethen also evaluated with the diagnostic method.

Yet another aspect of the invention is to use two different diagnosticmethods to eliminate the possibility that a substance used in thediagnostic test itself is having an effect on aspirations ofgastro-intestinal contents into the respiratory tract.

Another aspect of the invention is that if one of the substances used asa diagnostic test is found to be reducing or eliminating theaspirations, then that substance can be used as the agent to minimize orprevent aspirations.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the methods and formulations as more fully described below.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and formulations are described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aparticle” includes a plurality of such particles and reference to “thelabel” includes reference to one or more labels and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The invention includes different methods for diagnosing respiratoryfluid in a subject wherein the respiratory fluid has been regurgitatedfrom the gastrointestinal tract from the subject. The method includesorally administering to a subject a formulation comprised of a pluralityof particles which particles are comprised of a biocompatible materialsuch as a biocompatible polymer or a wax such as a caruba wax whereinthe particles include some type of detectable lable which may be afluorescent label, a radioactive label, a magnetic label or a UVdetectable label. The formulation is allowed to remain in the subjectover a period of time during which the subject would be expected toaspirate the formulation from the gastrointestinal tract. For example,the subject might be administered the formulation just prior to going tosleep. After allowing for sufficient time respiratory fluid is extractedfrom the patient. That respiratory fluid is analyzed in order todetermine if it contains the detectable label.

Once the patient has been determined as aspirating fluid from thegastrointestinal tract into the respiratory tract the patient willrequire treatment. That treatment involves orally administering to thesubject a formulation which comprises a pharmacological substance. Thatpharmacological substance or pharmaceutically active drug isadministered in order to reduce or prevent aspiration of fluid from thegastrointestinal tract into the respiratory tract. The pharmaceuticallyactive drug is preferably a drug which acts locally on thegastrointestinal tract and is not absorbed systemically. Examples ofsuch drugs are drugs selected from the group consisting of cromolynsalts, cromolynic acid, nedocromil, nedocromil salts or muscarinic acidreceptor antagonist.

Dosing of the drug to the patient will vary depending on a wide range offactors including the patient's age, size, weight, sex and condition.However, dosing of the drug to the patient is generally carried out byoral administration of the drug in the form of pills, capsules orsolutions. Pills and capsules may contain the drug in combination with apharmaceutically acceptable excipient. Solutions or suspensions maycontain the drug in an aqueous solution or suspension withpharmaceutically acceptable carriers.

The oral formulation may be administered to the subject just prior togoing to bed at night. Further, additional doses may be administeredduring the night depending on the subjects responsiveness to themedication. The dose may be administered before and/or after an activitysuch as going to sleep which is likely to result in aspirations. Thatactivity can be going to sleep for the evening, taking an afternoon nap,or after eating a large meal or drinking heavily.

In one embodiment of the invention patients undergoing lung transplantsare treated prophylactically in order to reduce or prevent intestinalfluid into the newly transplanted lung.

The dosing amount will also vary with the particular drug. Whenadministering cromolyn and in particular cromolyn salts currentlymarketed safe dosages for children for other indications have been shownto be in the range of about 20 mg 4 times per day to 40 mg 4 times perday. Adults have been dosed in the amount of 200 mg 4 times per day to400 mg 4 times per day.

The formulation may include both an immediate release component where adrug is immediately released and a controlled release component wherethe drug is not released immediately (e.g. over the first hour) butreleased gradually during hours 2 to about 8 hours after administration.Oral liquid formulations can be viscous formulations which provide adegree of coating to the gastrointestinal tract.

When administering cromolyn in order to carry out diagnostics thecromolyn should be delivered with significant amount of water, e,g. 6 ozor more, 12 oz or more, 16 oz or more of water. However, when thecromolyn is being administered in order to treat the subject it ispreferably delivered in the absence of water or with a very small amountof water e.g. 4 oz or less, 2 oz or less, 1 oz or less.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Fluorescein-Spray Drying

Prepare nuclei of <1 micron fluorescein particles by spray dryingaqueous solutions of fluorescein. Then condense vapors ofrespiratory-tract compatible waxes such as carnauba was upon thefluorescein particles completely encapsulating the fluorescein. Make asuspension of these particles in water using usual pharmaceuticalmethods to stabilize these, add flavor etc. Subject swallows a preciseamount of the liquid suspension prior to activity that is causing GITreflux (e.g., prior to going to sleep).

The health care provider takes a sample of airway fluid through inducedcoughing, bronchoscopy, spontaneous coughing etc. The sample may bediluted in additional water, or a solvent that dissolves the wax. Thefluorescent label is then released either as a result of the addition ofa suitable solvent, or by increasing the temperature to dissolve thewax, or both.

The concentration of material in the respiratory tract entering due toreflux is estimated from the intensity of fluorescence using one of manydetectors for fluorescence. The important parameter is the concentrationof the fluorescent label per volume of the airway fluid in which it wascontained as that is likely to be related to the harmful effects of thegastrointestinal contents in the respiratory tract.

Example 2 Fluorescein-Flow Focusing

Prepare nuclei of <1 micron fluorescein particles by extruding abiocompatible wax (carnauba) in an outer tube and a fluorescein label inan inner tube in order to completely encapsulate the fluorescein.Details of the flow focusing method are described in U.S. Pat. No.6,116,516 and related issued patents, all of which are incorporatedherein by reference. Make a suspension of these particles in water usingusual pharmaceutical methods to stabilize these, add flavor etc. Subjectswallows a precise amount of the liquid suspension prior to activitythat is causing GIT reflux (e.g., prior to going to sleep).

The health care provider takes a sample of airway fluid through inducedcoughing, bronchoscopy, spontaneous coughing etc. The sample may bediluted in additional water, or a solvent that dissolves the wax. Thefluorescent label is then released either as a result of the addition ofa suitable solvent, or by increasing the temperature to dissolve thewax, or both.

The concentration of material in the respiratory tract entering due toreflux is estimated from the intensity of fluorescence using one of manydetectors for fluorescence. The important parameter is the concentrationof the fluorescent label per volume of the airway fluid in which it wascontained as that is likely to be related to the harmful effects of thegastrointestinal contents in the respiratory tract.

Example 3 Magnetic Particles-Flow Focusing

Magnetic particles may be suspended in a formulation and then swallowedfor the diagnostic purposes described in this invention. However, it maybe desirable to protect these particles from digestion in thegastrointestinal tract. Further, unencapsulated magnetic particles couldbe harmful to either the gastrointestinal tract, or the respiratorytract, or both. Using the flow focusing method it is possible tomanufacture biocompatible encapsulated magnetic particles that are notdigested in the gastrointestinal tract. Prepare nuclei of <1 micronmagnetic particles by extruding a biocompatible wax (carnauba) in anouter tube and a magnetic particle label in an inner tube in order tocompletely encapsulate the magnetic particle. Make a suspension of theseparticles in water using usual pharmaceutical methods to stabilizethese, add flavor etc. Subject swallows a precise amount of the liquidsuspension prior to activity that is causing GIT reflux (e.g., prior togoing to sleep).

The health care provider takes a sample of airway fluid through inducedcoughing, bronchoscopy, spontaneous coughing etc. The concentration ofthe gastrointestinal contents in the respiratory tract can be estimatedby collecting with a magnet the magnetic particles and then countingthem using one of the many methods available for such counting, or bymeasurement of the total magnetism. The sample may be also diluted inadditional water, or a solvent that dissolves the wax. The magneticparticles can then be released either as a result of the addition of asuitable solvent, or by increasing the temperature to dissolve the wax,or both.

The important parameter is the concentration of the magnetic particlelabel per volume of the airway fluid in which it was contained as thatis likely to be related to the harmful effects of the gastrointestinalcontents aspirated into the respiratory tract.

Example 4 UV Labeled Particles-Flow Focusing

Prepare nuclei of <1 micron UV labeled particles by extruding abiocompatible wax (carnauba) in an outer tube and a UV label or UVlabeled particle in an inner tube in order to completely encapsulate theUV labeled particle. Make a suspension of these particles in water usingusual pharmaceutical methods to stabilize these, add flavor etc. Subjectswallows a precise amount of the liquid suspension prior to activitythat is causing GIT reflux (e.g., prior to going to sleep).

The health care provider takes a sample of airway fluid through inducedcoughing, bronchoscopy, spontaneous coughing etc. The sample may bediluted in additional water, or a solvent that dissolves the wax. The UVlabeled particle is then released either as a result of the addition ofa suitable solvent, or by increasing the temperature to dissolve thewax, or both.

The amount of material in the respiratory tract entering due to refluxis estimated from the UV labeled particles detected using standarddetectors. The important parameter is the concentration of the UVlabeled particles per volume of the respiratory fluid in which it wascontained as that is likely to be related to the harmful effects of thegastrointestinal contents aspirated into the respiratory tract.

Example 5 Phosphorescent Particles-Flow Focusing

Prepare nuclei of <1 micron phosphorescent labeled particles byextruding a biocompatible wax (carnauba) in an outer tube and aphosphorescent labeled particle in an inner tube in order to completelyencapsulate the phosphorescent labeled particle. Make a suspension ofthese particles in water using usual pharmaceutical methods to stabilizethese, add flavor etc. Subject swallows a precise amount of the liquidsuspension prior to activity that is causing GIT reflux (e.g., prior togoing to sleep).

The health care provider takes a sample of airway fluid through inducedcoughing, bronchoscopy, spontaneous coughing etc. The sample may bediluted in additional water, or a solvent that dissolves the wax. Thephosphorescent labeled particle is then released either as a result ofthe addition of a suitable solvent, or by increasing the temperature todissolve the wax, or both.

The amount of material in the respiratory tract entering due to refluxis estimated from the phosphorescent labeled particles detected usingstandard detectors. The important parameter is the concentration of thephosphorescent labeled particles per volume of the airway fluid in whichit was contained as that is likely to be related to the harmful effectsof the gastrointestinal contents aspirated into the respiratory tract.

Example 6 Fluorescein-Flow Focusing with Cromolyn Sodium)

Prepare nuclei of <1 micron fluorescein particles by extruding abiocompatible wax (carnauba) in an outer tube and a fluorescein label inan inner tube in order to completely encapsulate the fluorescein. Make asuspension of these particles in water using usual pharmaceuticalmethods to stabilize these, dissolve cromolyn sodium and add flavor etc.Subject swallows a precise amount of the liquid suspension including thedissolved cromolyn sodium prior to activity that is causing GIT reflux(e.g., prior to going to sleep).

The health care provider takes a sample of airway fluid through inducedcoughing, bronchoscopy, spontaneous coughing etc. The sample may bediluted in additional water, or a solvent that dissolves the wax.Fluorescein is then released either as a result of the addition of asuitable solvent, or by increasing the temperature to dissolve the wax,or both.

If the carnauba wax particles were suspended in a solution of cromolyn,blood or urine samples are also taken and checked for cromolyn toestimate the total amount of gastrointestinal contents that entered therespiratory tract. The amount of cromolyn in the blood or urine showshow much of the formulation of cromolyn swallowed did enter therespiratory tract, because cromolyn will not enter the blood stream, orurine,via the GI tract.

The fluorescein particles encapsulated in carnauba wax enter therespiratory tract if the subject aspires the contents of her/hisgastrointestinal tract. Fluorescein can be released from the carnaubawax particles present in the respiratory fluid by heating or dissolvingthe wax using organic solvents. The concentration of material in therespiratory tract entering due to such aspiration is estimated using oneof many methods to detect and quantify fluorescein. The importantparameter is the concentration of fluorescein per volume of the airwayfluid in which it was contained as that is likely to be related to theharmful effects of the gastrointestinal contents aspirated into therespiratory tract. The presence of cromolyn in a body fluid such asplasma, serum or preferably urine can be quantified by a variety ofmethods used to measure cromolyn concentrations, such as HPLC with asuitable detector or radioimmunoassay. Measurement of samples of bodyfluid enables to estimate the total amount of cromolyn that entered therespiratory tract, and therefore provides an estimate of the totalamount of gastrointestinal fluid that entered the patient's respiratorytract. Therefore, the combination of the two diagnostic agents providesestimates of both the concentration of the gastrointestinal fluid in therespiratory tract as well as the total amount of the contents ofgastrointestinal fluid that entered the respiratory tract.

Example 7 Treatment of Aspirations of Gastrointestinal Contents into theRespiratory Tract

The subject swallows prior to going to bed an aqueous suspension ofcarnauba wax particles that contain encapsulated fluorescein. Thesubject then collects any sputum that has been spontaneously producedovernight. If insufficient sputum is obtained, the subject isadministered a mist of hypertonic saline by inhalation to induce sputumproduction. The sputum sample is diluted with a high pH buffer andorganic solvent immiscible with water is added to extract the wax. Theaqueous phase is separated and a sample is analyzed for fluorescenceusing a fluorescence detector such as a fluorimeter. If the fluorescenceintensity exceeds the limit previously established for healthy subjects,it is assumed that the subject who has just undergone the test suffersfrom aspirations of gastrointestinal contents into the respiratorytract. The test may need to be repeated several times in case theaspirations do not occur every night, especially if the subject alreadyhas a condition that is suspected to cause such aspirations, or thesubject has respiratory symptoms of aspirations. When the diagnosis isconfirmed, the subject takes (prior to going to bed) a dose of cromolynsodium and collects urine samples overnight as well as the first thingin the morning. The subject then either sends the urine sample foranalysis of cromolyn to a laboratory, or uses a dipstick test at home.The subject repeats the test for several days. If the amount of cromolyndetected in the urine does not exceed the amounts typically found insubjects without aspirations, or the cromolyn concentration in the urinecontinues to get smaller upon successive testing, it may be concludedthat cromolyn (in this subject) is effective to minimize or preventaspirations of gastric contents into the respiratory tract and it willbe therefore used for this purpose for as long as the condition exists,or the treatment ceases to be effective.

Example 8 Treatment of Aspirations of Gastrointestinal Contents into theRespiratory Tract

The subject swallows prior to going to bed an aqueous suspension ofcarnauba wax particles that contain encapsulated fluorescein. Thesubject then collects any sputum that has been spontaneously producedovernight. If insufficient sputum is obtained, the subject isadministered a mist of hypertonic saline by inhalation to induce sputumproduction. The sputum sample is diluted with a high pH buffer andorganic solvent immiscible with water is added to extract the wax. Theaqueous phase is separated and a sample is analyzed for fluorescenceusing a fluorescence detector such as a fluorimeter. If the fluorescenceintensity exceeds the limit previously established for healthy subjects,it is assumed that the subject who has just undergone the test suffersfrom aspirations of gastrointestinal contents into the respiratorytract. When the diagnosis is confirmed, the subject takes (prior togoing to bed) carnauba wax particles that contain encapsulatedfluorescein, suspended in an aqueous solution of cromolyn sodium. Thesubject then collects a sputum sample in the morning, or goes to ahealthcare professional who will collect a sample of the respiratoryfluid which is then analyzed for the presence of the fluorescein. If thetest is negative, the subject may need to repeat for several days toconfirm that the cromolyn prevents or reduces the aspiration ofgastrointestinal fluid into the respiratory tract.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

That which is claimed is:
 1. A method of treating a subject sufferingfrom entry of gastrointestinal contents into the respiratory tract,comprising: orally administering to a subject a diagnostic formulationcomprising an agent that is not absorbed from the gastrointestinal tractof a mammal but is absorbed from the respiratory tract of a mammal;allowing the diagnostic formulation to remain in the subject over aperiod of time during which the subject would be expected to aspiratethe formulation from the gastrointestinal tract into the respiratorytract; accessing a body fluid from the subject, wherein the body fluidis urine, blood or a blood product; analyzing the body fluid to detectthe level of the agent in the body fluid; determining that the fluidcontains a level of the agent indicative of aspiration ofgastrointestinal contents into the respiratory tract, and providing anintervention to prevent or reduce such aspirations in the subject. 2.The method of claim 1, wherein the body fluid is collected prior toanalyzing the body fluid.
 3. The method of claim 1, wherein theadministered agent is selected from the group consisting of cromolynsalts, cromolynic acid, nedocromil, nedocromil salts and muscarinic acidreceptor antagonists.
 4. The method of claim 1, wherein the administeredagent is a cromolyn salt.
 5. The method of claim 1, further comprising:administering a control formulation comprising the same agent as foundin the diagnostic formulation to the subject at an occasion different tothat when the orally administered diagnostic formulation isadministered; accessing a body fluid from the subject following theadministration of the control formulation; analyzing the body fluid todetermine if the fluid contains the agent; and determining that thelevel of agent detected following administration of the diagnosticformulation is indicative of a disease associated with aspiration ofgastrointestinal contents into the respiratory tract by comparison tothe level of agent detected following administration of the controlformulation.
 6. The method of claim 5, wherein the control formulationis administered orally.
 7. The method of claim 5, wherein the controlformulation is administered by inhalation.
 8. The method of any of claim5, wherein the body fluid is collected following the administration ofthe control formulation and prior to analyzing the body fluid.
 9. Themethod of claim 5, wherein the control formulation is administeredduring a period of time when the subject is not expected to experienceaspiration of gastrointestinal contents into the respiratory tract. 10.The method of claim 1, further comprising: detecting a level of theagent indicative of aspiration of gastrointestinal contents into therespiratory tract based on the level of the agent observed followingadministration of the control formulation; and administering to thesubject a medical treatment to reduce or prevent such aspiration.
 11. Amethod of treating a subject suffering from entry of gastrointestinalcontents into the respiratory tract, comprising: orally administering toa subject a diagnostic formulation comprising a cromolyn salt; allowingthe diagnostic formulation to remain in the subject over a period oftime during which the subject would be expected to aspirate theformulation from the gastrointestinal tract into the respiratory tract;accessing a body fluid from the subject, wherein the body fluid isurine, blood or a blood product; analyzing the body fluid to detect thelevel of cromolyn salt in the body fluid; determining that the fluidcontains a level of cromolyn salt indicative of aspiration ofgastrointestinal contents into the respiratory tract, and providing anintervention to prevent or reduce such aspirations in the subject. 12.The method of claim 11, wherein the body fluid is collected prior toanalyzing the body fluid.
 13. The method of claim 11, furthercomprising: administering a control formulation comprising the cromolynsalt found in the diagnostic formulation to the subject at an occasiondifferent to that when the orally administered diagnostic formulation isadministered; accessing a body fluid from the subject following theadministration of the control formulation; analyzing the body fluid todetermine if the fluid contains the cromolyn salt; and determining thatthe level of cromolyn salt detected following administration of thediagnostic formulation is indicative of a disease associated withaspiration of gastrointestinal contents into the respiratory tract bycomparison to the level of cromolyn salt detected followingadministration of the control formulation.
 14. The method of claim 13,wherein the control formulation is administered orally.
 15. The methodof claim 13, wherein the control formulation is administered byinhalation.
 16. The method of claim 13, wherein the body fluid iscollected following the administration of the control formulation andprior to analyzing the body fluid.
 17. The method of claim 13, whereinthe control formulation is administered during a period of time when thesubject is not expected to experience aspiration of gastrointestinalcontents into the respiratory tract.
 18. The method of claim 11, furthercomprising: detecting a level of the agent indicative of aspiration ofgastrointestinal contents into the respiratory tract based on the levelof the agent observed following administration of the controlformulation; and administering to the subject a medical treatment toreduce or prevent such aspiration.
 19. A dipstick device for thedetection of an agent indicative of aspiration of gastrointestinalcontents into the respiratory tract of a subject, comprising: asubstrate comprised of paper and a first reagent that reacts with theagent; and a second reagent that enables the reaction of the firstreagent to form a dye with the agent that is of a different color thanthe color either the first or the second reagent.
 20. The method ofclaim 19, wherein the agent is selected from the group consisting ofcromolyn salts, cromolynic acid, nedocromil, nedocromil salts andmuscarinic acid receptor antagonists.
 21. The method of claim 19,wherein the agent is a cromolyn salt.
 22. A dipstick device for thedetection of an agent indicative of aspiration of gastrointestinalcontents into the respiratory tract of a subject, comprising,comprising: a substrate comprised of paper and a reagent that reactswith the agent to form a compound with a visually detectable color. 23.The method of claim 22, wherein the agent is selected from the groupconsisting of cromolyn salts, cromolynic acid, nedocromil, nedocromilsalts and muscarinic acid receptor antagonists.
 24. The method of claim22, wherein the agent is a cromolyn salt.
 25. A dipstick device for thedetection of an agent indicative of aspiration of gastrointestinalcontents into the respiratory tract of a subject, comprising: asubstrate comprised of paper and a first reagent that reacts with theagent; and a second reagent that enables the reaction of the firstreagent to form a compound with a visually detectable color.
 26. Themethod of claim 25, wherein the agent is selected from the groupconsisting of cromolyn salts, cromolynic acid, nedocromil, nedocromilsalts and muscarinic acid receptor antagonists.
 27. The method of claim25, wherein the agent is a cromolyn salt.